In-Gel Sample Digestion Protocol

Reagents required

Reagent Setup (prepare freshly before use)

Procedure

1. Wipe the glass slides, scalpel with methanol. Excise the protein band from acrylamide gel with the scalpel on a glass slide avoiding the unstained gel region.
2. Chop excised bands into small cubes of 1 x 1 mm size and transfer the pieces into duly labelled 1.5 ml centrifuge tubes.



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3. Rinse the gel bands with milliQ water for 1 hour by vortexing @1500rpm
4. Discard the water and repeat the rinsing step for another 1 hour with 1 ml of fresh milliQ water.
5. Spin gel cubes at 3000 rpm for 20 seconds and remove the water completely.
6. Add 300 μl of destaining solution and vortex for 30 minutes @1500 rpm, depending on the staining intensity.
7. Add 500 μl of 100% acetonitrile and incubate at room temperature with vortexing for 10-15 minutes or until the gel pieces become white and shrink.
8. Remove acetonitrile completely and keep the vials open for 20 minutes for the solvent to evaporate completely.
9. Add 30-50 μl of 13 ng/μl trypsin buffer in such a way that it covers the dry gel pieces and leave it in an ice bucket or a fridge for saturating the gel.
10. After 30 minutes, check the level of trypsin buffer and if necessary, add more trypsin buffer for the gel pieces to be completely covered with trypsin buffer during the digestion step.
11. Leave the gel pieces for another 90 minutes to saturate them with trypsin buffer. Check the levels of trypsin buffer in the vials again to see if the gels are remaining submerged. If needed, add 10-20μl of ammonium bicarbonate buffer so that the gel pieces remain wet during enzymatic cleavage..
12. Incubate the vials at 37°C at 300 rpm overnight with gentle vortexing.
13. Remove the trypsin buffer from the sample vials to respective freshly labelled 0.6 ml of microcentrifuge tubes.
14. Add the extraction buffer (twice the volume of trypsin buffer used initially, usually 100μl) to the sample tubes containing gel pieces and vortex vigorously for 20 minutes.
15. Aspirate the extraction buffer and combine it with the 0.6 ml microcentrifuge tubes.
16. Dry the entire samples using Speed Vacuum concentrator and store at -20 until use.
17. Finally reconstitute the samples in 30 μl of 3% acetonitrile before the LC/MS analysis.

In-Solution Sample Preparation Guidelines

The quality of sample extraction and preparation significantly impact MS results. For in-solution samples, strictly follow the following guidelines:

• DO NOT wash any flasks, tubes, or glass plates (for sample preparation) with soap (or any polymeric detergent). Always, rinse your glassware with hot water and then an organic solvent like 70% ethanol or methanol.
• Freshly prepare all the buffers for MS analysis.
• CRITICAL: : Mass spectrometry is highly sensitive to various contaminants, such as PEG (polyethylene glycol), keratin and various salts. Therefore, it is recommended to use powder free nitryl gloves and work in a dust- free environment.
• CRITICAL: The preferred MS compatible protein extraction buffer, ideal for optimum trypsin activity, is 50 mM ammonium bicarbonate buffer. If you had used any other buffer for washing or re-suspending the cell pellets (or tissues), desalting (or dialysis) and buffer exchange (using 50 mM ammonium bicarbonate) should be performed. It can be accomplished using a centrifugal filter with an appropriate molecular-weight-cut-off (MWCO) membrane such as Amicon Ultra 0.5 mL centrifugal filters having 3 kDa MWCO (P/N : UFC500324, Merk Millipore). Please supply the protein sample in 50 mM ammonium bicarbonate buffer.
• CRITICAL: The commonly used detergents are NOT compatible with MS. These include NP-40, Triton, CHAPS, SDS (sodium dodecyl sulfate) and LDS (Lithium dodecyl sulfate), Octyl glucoside, and octyl thioglucoside, sodium deoxycholate, lauryl maltoside, Brij-35, etc. If you have to use detergent for the cell lysis, use only MS-compatible detergents. A few MS-compatible detergents are listed below.
  • RapiGest SF from Waters
    https://www.waters.com/nextgen/in/en/shop/standards--reagents/186001861-rapigest-sf-1-mg--5-pk.html
  • PPS Silent Surfactant from Agilent
    https://www.chem-agilent.com/pdf/strata/400500.pdf
  • Detergent-free buffer from Sigma
    https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/protein-biology/protein-lysis-and-extraction/extraction-from-tissue
• After desalting and buffer exchange, do protein quantification. The sample (protein) has to have a concentration of 1 mg/mL and 100 microlitres is required (100 micrograms in 100 microlitres). If the concentration of your sample is less than 1 mg/mL, let us know in advance.
• If you have samples to be compared (for relative protein quantification), all samples should be in uniform concentration and volume.
• We suggest sharing the protein isolation protocol (which you intend to follow) with the facility staff and getting their approval before proceeding with the sample preparation.
• Shipment:The samples need to be shipped in a frozen condition in dry ice or brought to RGCB in ice by hand.

In-Solution Sample Digestion Protocol

Reagents Required

Reagent Setup (prepare freshly before use)

1. DTT solution: 100 mM DTT in 50 mM ammonium bicarbonate.
2. Iodoacetamide solution: 200 mM Iodoacetamide solution in 50 mM ammonium bicarbonate.
3. Formic acid 1% in milliQ water (v/v).
4. Trypsin buffer: 0.4 μg /μl trypsin in 50 mM ammonium bicarbonate.

Trypsin digestion protocol for 100 μg of protein sample

1. Add 5μl of 100 mM DTT solution to the protein sample.
2. Incubate at 60° Celsius for 30 minutes
3. Add 5 μl of 200 mM Iodoacetamide solution to the protein sample.
4. Incubate at room temperature in dark for 30 minutes.
5. Add trypsin buffer in the ratio of 1:25 (1μg of trypsin to 25μg of protein).
6. Incubate at 37° Celsius overnight with gentle shaking.
7. To arrest the reaction, add 1 μl of 1% formic acid (v/v) to the samples.
8. Incubate at 37° Celsius for 20 minutes.
9. Store the samples at -20 deep freezer until use.